Accelerated graft arteriosclerosis (AGA) is the most common long-term cause of death in patients with cardiac allografts. AGA is characterized by a neointima of smooth muscle cells and macrophages in the arteries of transplanted organs. Although the pathogenesis of AGA is not completely understood, a variety of inducers--including complement, granzyme B, antibodies, and viral infections--may play a role in the process of inflammation and proliferation that ultimately leads to AGA. We and others have previously shown that nitric oxide (NO) inhibits AGA. In particular, NO derived from the inducible NO synthase (iNOS, or NOS2) appears to limit the vasculopathy that is a hallmark of AGA. For example, adenoviral delivery of NOS2 decreases AGA. In contrast, inhibitors of NOS increase AGA. Furthermore, genetic deletion of NOS2 exacerbates AGA: cardiac allografts develop more severe AGA when transplanted into NOS2 knockout mice, compared to hearts transplanted into wild-type mice. Thus NO derived from NOS2 protects cardiac allografts from AGA. The molecular mechanisms by which NO inhibits AGA are unknown. However, we recently discovered that NOS2 decreases inflammation in cardiac allografts. In particular, we found that NOS2 inhibits the release ot Weibel-Palade bodies from endothelial cells in donor hearts. Since Weibel-Palade bodies contain inflammatory and thrombotic mediators, inhibition of Weibel-Palade body release may explain part of the anti-inflammatory effects of NO in AGA and other vascular diseases. We now propose to explore the molecular mechanism by which NO inhibits Weibel-Palade body release. Preliminary Data shows that NO blocks Weibel-Palade body release from cultured endothelial cells. We will begin by determining whether or not NO can inhibit the triggering of Weibel-Palade body release by various inducers of AGA. Then we will define the mechanisms by which Weibel-Palade bodies are normally released. We will next determine the molecular targets of NO. Finally, we will examine the physiological relevance of these mechanisms in a murine model of AGA. These studies will characterize novel molecular mechanisms by which radicals regulate vascular inflammation.